Bioprotectant composition, method of use and extraction process of curcuminoids

ABSTRACT

PCT No. PCT/US96/11431 Sec. 371 Date Oct. 27, 1997 Sec. 102(e) Date Oct. 27, 1997 PCT Filed Jul. 12, 1996 PCT Pub. No. WO97/03674 PCT Pub. Date Feb. 6, 1997Curcuminoids have been found to have anti-oxidant, anti-inflammatory, antibacterial, antifungal, antiparasitic, anti-mutagen, anticancer and detox properties. The present invention is directed to compositions containing three curcuminoids, i.e. curcumin, demethoxy curcumin and bis demethoxy curcumin, extracted from roots of tumeric. These compositions have been found to have activity in the anti-oxidant mechanisms of prevention and intervention.

This is a prov. appln. of Ser. No. 60-001,161, filed Jul. 14, 1995.

I. A novel concept of bioprotectant activity

Curcuma longa (Fam. Zingiberaccae) or Turmeric is one of the oldestherbs in Ayurveda materia medica, and has been used in Ayurveda medicineinternally as a stomach, tonic and blood purifier, and topically in theprevention and treatment of skin diseases. The significance of turmericin medicine has changed considerably since the very recent discovery ofthe anti-oxidant properties of naturally occurring phenolic compounds.The same ground dried rhizome of Curcuma longa, which has been used forcenturies as a spice, food preservative and a coloring agent, has beenfound to be a rich source of phenolic compounds or curcuminoids. Thereare three main curcuminoids recognized, i.e.,curcumin(diferuloylmethane), demethoxy curcumin(p-hydroxycinnamoylferuloyl!methane) and bis demethoxycurcumin(p,p-dihydroxydicinnamoylmethane).

Curcuminoids have scientifically documented anti-oxidant,anti-inflammatory, anti-bacterial, anti-fungal, antiparasitic,anti-mutagen, anti-cancer and detox properties. Their potential use inthe prevention of cancer and in the treatment of infection with humanimmunodeficiency virus (HIV) are the subject of intensive laboratory andclinical research. Curcuminoids are recognized for their broadbiological activity and safety of use. The biological activity ofcurcuminoids can best be described by the word "protective".

Curcumin and turmeric have been used as anti-inflammatory drugs for manyyears. More recently, there has been much interest in possiblechemopreventive effects of curcumin. Curcumin inhibits tumor initiationby benxo a!pyrene and tumor promotion by12-0-tetradecanoylphorbol-13-acetate (TPA) in female CD-1 mice.

    ______________________________________    THE BIOLOGICAL ACTIVITIES OF CURCUMIN    The biological activity Inhibition    ______________________________________    1.      TPA-induced epidermal ornithine                                ++++            decarboxylase activity    2.      TPA-induced edema of mouse ear                                ++++    3.      TPA-induced epidermal hyperplasia                                ++++    4.      TPA-induced epidermal DNA                                +            synthesis    5.      Arachidonic acid-induced edema of                                +++            mouse ear    6.      Epidermal lipoxygenase                                ++++            activity    7.      Epidermal cyclooxygenase activity                                ++++    8.      TPA-induced skin tumor promotion                                ++++            in previously initiated mice    ______________________________________

Co-application of curcumin with TPA inhibits TPA-induced skininflammation, increased epidermal ornithine decarboxylase activity.Huang et al. have also shown that 2; curcumin in the diet inhibitsazoxymethane-induced foci in the colon of CF-1 mice and inhibitsduodenal tumorigenesis in C57BL/6J mice previously initiated withN-ethyl-N'-nitro-N-nitrosguanidine.

Many of the anti-inflammatory and chemopreventive properties of curcuminmay be related to the antioxidant properties of curcumin. In the presentstudy, the antioxidant properties of curcumin, demethoxycurcumin,bisdemethoxycurcumin and several preparations containing variedproportions of curcumin, demethoxycurcumin, bisdemethoxycurcumin weremeasured by the Rancimat method.

    ______________________________________     ##STR1##    R.sub.1     R.sub.2    ______________________________________    OCH.sub.3   OCH.sub.3  Curcumin    H           OCH.sub.3  Demethoxycurcumin    H           H          Bisdemethoxycurcumin    ______________________________________

The Rancimat method measures the conductivity changes caused byformation of small fatty acid molecules when the fats and oils areoxidized under elevated temperature and accelerated aeration. Theinduction times of lard with curcuminoids added were measured. Thelonger induction times suggest stronger antioxidant activity. Allcurcuminoids showed excellent antioxidant activity.

In chronic inflammation, cytokines induce the production of nitric oxidethat is converted to DNA damaging and carcinogenic peroxynitrite andnitrite. The effect of curcumin on the generation of peroxynitriteradicals and nitrite was studied. Curcumin inhibited lipopolysaccharide(LPS) and interferon γ (INFγ) induced nitrite production by mouseperitoneal cells by more than 50% at 2.5-10 uM (FIG. 6).

The present invention is directed to a composition containing threecurcuminoids, i.e., curcumin, demethoxy curcumin and bis demethoxycurcumin, extracted from roots of turmeric. Curcuminoids possessdistinct mechanisms which may explain their "protective" qualities. Thisprotective activity has been defined by testing one of the best knownproperties of curcuminoids, their anti-oxidant properties. Two distinctmodes of anti-oxidant action have been identified in curcuminoids: 1)prevention mode (Table 1) i.e. prevention of the formation of freeradicals, and 2) intervention mode (Table 2) i.e. neutralizing actionupon the already formed free radicals by the process of free-radicalscavenging (FIGS. 4 and 5).

                  TABLE 1    ______________________________________    Prevention mode - the bioprotectant    preventive activity of various compounds, as measured by    the rancimat method  The rancimat method measures the time    needed for inducing oxidative changes in the substrate -    the longer the "induction time" the stronger the anti-    oxidant properties!. Tested compounds were used in a    standard concentration of 0.02%                            INDUCTION TIME    SAMPLE                  IN HOURS    ______________________________________    CONTROL                 2.06    BHT (common synthetic phenolic food additive)                            5.05    CURCUMINOID COMPLEX Lot #4226                            6.05    GRAPE SEED EXTRACT Lot #3034                            2.13    PINE BARK EXTRACT Lot #1372                            2.13    ______________________________________

                  TABLE 2    ______________________________________    Intervention mode - the bioprotectant    intervention activity of curcuminoids, measured as DPPH    free-radical scavenging ability against a control with "0"    ability to scavenge free radicals.                 CONCENTRATION SCAVENGING    SAMPLE       ug/ml         ABILITY %    ______________________________________    CURCUMINOID  4             16    COMPLEX LOT NO.    RD/CUR/02    CURCUMINOID  8             31.4    COMPLEX LOT NO.    RD/CUR/02    CURCUMINOID  12            47.7    COMPLEX LOT NO.    RD/CUR/02    CURCUMINOID  20            73.8    COMPLEX LOT NO.    RD/CUR/02    ______________________________________

The composition of the present invention showed significant activity inboth anti-oxidant mechanisms studied (prevention and intervention). Itshould be noted that some other natural anti-oxidants, which have beenscreened together with curcuminoids, do not offer combined preventionand intervention activity. It has been concluded that the broadbiological mechanism of curcuminoids is owed, to a large extent, to thecombined mechanisms of prevention and intervention. Prevention andintervention result in the totality of protective qualities. Sincecurcuminoids may protect the integrity of biological systems bypreventing the free-radical assault and by intervening to stop theassault, it is proposed to classify curcuminoids and any similarcompound as a "bioprotectant".

The anti-oxidant potential of the composition was evaluated against theindividual curcuminoids, i.e., curcumin, demethoxy curcumin, bisdemethoxy curcumin, and other natural anti-oxidants like grape seedextract and pine bark extract in vitro. Interestingly, the presentcomposition showed better bioprotectant activities than did the purecompounds as well as the two other natural anti-oxidants.

    ______________________________________    THE OXIDATIVE INDUCTION TIME AND THE ANTIOXIDANT    INDEX OF LARD WITH AND WITHOUT ANTIOXIDANT ADDED                      Induction                               Antioxidant    Sample            time (Hrs)                               index    ______________________________________    Control           2.06    BHT (Synthetic phenolic)                      5.05     2.45    Curcumin 99% Lot #001                      2.83     1.37    BDM Curcumin Lot #06                      3.48     1.69    Curcuminoids Lot #4226                      6.05     2.92    Grape Seed Ex. Lot #3034                      2.13     1.03    Pine Bark Ex. Lot #1372                      2.13     1.03    ______________________________________

    ______________________________________    EFFECT OF CURCUMINOID COMPOSITION    ON THE ANTIOXIDANT INDEX IN RANCIMAT METHOD                      BDM        DM     Anti-             Curcumin Curcumin   Curcumin                                        oxidant    Sample   %        %          %      Index    ______________________________________    SAB 25   78.6     2.2        16.7   2.7    SAB 26   86.6     1.9        8.3    2.4    SAB 32   80.2     2.5        15.5   2.0    SAB 38   70.8     4.5        18.5   2.1    SAB 40   67.2     2.8        14.8   1.5    ______________________________________

II. The unique formula for a curcuminoid composition

It has been found that the composition of curcuminoids may affect theirbioprotectant activity, since individual components display differentbioprotectant potential. For example, curcumin alone does not provide astrong prevention against free-radicals. On the other hand, curcuminshows strong intervention properties in neutralizing the already formedfree-radicals. Similarly, a derivative of curcumin, tetrahydrocurcumin(THC), provides excellent intervention in scavenging already formedfree-radicals, but is considerably less potent in the prevention offree-radical formation.

As a result of experimentally confirmed differences among variouscurcuminoid combinations, it has been concluded that only a carefullybalanced composition of curcuminoids and derivatives thereof can resultin optimal bioprotectant activity.

                  TABLE 3    ______________________________________    Relationship between the composition of    curcuminoid mixtures and the anti-oxidant activity as    measured with the Rancimat method  The longer the    induction time the better the anti-oxidant activity!.                     DM       BDM           Induction    Tested  Curcumin Curcumin Curcumin                                     Total  Time    Compound            %        %        %      Curcumins                                            (hrs.)    ______________________________________    1. Control            none     none     none   none   2.00    2. Complex            78.6     16.7     2.2    97.5   5.55    3. Complex            72.0     19.4     6.7    98.4   5.32    4. Complex            73.9     18.1     3.4    95.4   5.28    5. Complex            75.1     17.8     2.3    95.2   5.20    6. Complex            74.2     20.6     3.1    97.9   5.08    7. Complex            72.9     18.9     4.7    96.5   5.07    8. Complex            86.6      8.3     1.9    96.8   4.80    ______________________________________

The following observations were made pertaining to the relationshipbetween the different compositions of curcuminoid combinations and theiranti-oxidant activity:

the mixture of curcuminoids is generally a more effective bioprotectantthan either of the three components alone;

the proportion of the individual components occurring in a mixture ofcurcuminoids is an important factor in determining the bioprotectantproperties;

the total content of curcuminoids is of secondary importance to thebioprotectant properties, primary importance being the presence of thethree curcuminoids in the appropriate weight ratio of the individualcomponents;

the addition of certain derivatives of curcuminoids, e.g.,tetrahydrocurcumin; cyclo curcumin; tumerin; curcumin complexed withmetals like potassium, zinc, calcium, copper, chromium, vanadium; etc.may increase the bioprotectant activity of curcuminoids.

The following composition of curcuminoids is preferred to afford maximumbioprotectant activity:

1. Curcumin should be present in an amount of no less than 75% and nomore than 81% of the total curcuminoids.

2. Demethoxy curcumin should be present in an amount of no less than 15%and no more than 19% of the total curcuminoids.

3. Bis demethoxy curcumin should be present in the amount of no lessthan 2.2% and no more than 6.5% of the total curcuminoids.

4. Additional ingredients may be present, e.g., tetrahydrocurcumin in anamount ranging from 1% to 5% ; curcuminoids complexed with metals likepotassium, zinc, copper, chromium, vanadium, calcium, etc. in an amountranging from 1% to 5% ; alkaloid piperine preferably in the form ofBioperine™ (subject of U.S. application Ser. No. 08/393,738 filed onFeb. 24, 1995) in an amount ranging from 0.001% to 1%; cyclo curcumin inan amount ranging from 1% to 5%; and tumerin in an amount ranging from0.1% to 0.5%.

The following are examples of preferred combinations of curcuminoids andother ingredients:

1. Curcumin 78.6%, demethoxy curcumin 16.7%, bis demethoxy curcumin2.5%, tetrahydrocurcumin 1%, potassium curcumin 0.5%, Bioperine™(piperine) 0.5%.

2. Curcumin 80.2%, demethoxy curcumin 15.5%, bis demethoxy curcumin2.5%, tetrahydrocurcumin 1.8%.

3. Curcumin 75%, demethoxy curcumin 15%, bis demethoxy curcumin 6.5%,tetrahydrocurcumin 2%, Bioperine™ (piperine) 1%.

4. Curcumin 76.8%, demethoxy curcumin 16.1%, bis methoxy curcumin 6.1%.

5. Curcumin 78.6%, demethoxy curcumin 16.7%, bis demethoxy curcumin2.5%, cyclo curcumin 1%, potassium curcumin 0.5%, Bioperine™ (piperine)0.5%.

6. Curcumin 78.6%, demethoxy curcumin 16.7%, bis demethoxy curcumin2.5%, tumerin 0.1%, potassium curcumin 0.5%, Bioperine™ (piperine) 0.5%.

7. Curcumin 80.2%, demethoxy curcumin 15.5%, bis demethoxy curcumin2.5%, cyclo curcumin 1.8%.

8. Curcumin 75%, demethoxy curcumin 15%, bis demethoxy curcumin 6.5%,cyclo curcumin 2%, Bioperine™ (piperine) 1%.

The present invention is the optimal composition showing both preventionand intervention activity according to the above provided definition ofbioprotectants.

III. The unique manufacturing procedure

The specific combination of the ingredients in the preparation can beaccomplished by way of a novel extraction procedure of curcuminoids fromrhizomae of Curcuma longa. The curcuminoid combination can be isolatedby:

a) drying and powdering tumeric rhizomae to produce a resulting powder,

b) extracting the resulting powder with a solvent at a temperaturebetween 30°-60° C. to produce an extract,

c) concentrating said extract,

d) lowering the temperature of said concentrated extract to between0°-15° C. to crystallize any curcuminoids present,

e) isolating any resulting crystals,

f) dissolving any isolated crystals of curcuminoids in a solvent at atemperature between 30°-50° C.,

g) lowering the temperature of the dissolved curcuminoids to atemperature between 0°-15° C., and

h) isolating any curcuminoids present.

Examples of suitable solvents used to extract the powder are ethylenedichloride, methylene dichloride and ethyl acetate. The volume ofsolvent can be 3to 9 volumes calculated on the dry weight of powderedturmeric rhizomae. The extraction procedure can be repeated severaltimes and the individual extracts combined before concentration. Theextracts can be filtered and concentrated by distillation under vacuumat temperatures just under 50° C. Suitable solvents for dissolving thecrystals of curcuminoids are C₁ -C₆ alkyl ketone solvents preferablyacetone, methyl ketone, etc. Steps d)-g) are critical for obtaining thedesired composition of curcuminoids. As a result of this extractionprocedure the unique composition of curcuminoids is obtained.

Characteristics of the composition of the invention

Description: orange yellow crystalline powder.

Solubility: slightly soluble in alcohol, soluble in acetone and inglacial acetic acid.

Identification: by UV absorption; by boric acid test--dilute ethanolicsolution after acidifying with hydrochloric acid gives a reddish colorwith boric acid.

Melting range: melts between 180° and 185° C.

Loss on drying: not more than 0.5% w/w.

Assay by HPLC: determines composition of curcuminoids within specifiedrange.

B. The method for complexation of curcuminoids with metals is also anovel procedure. Curcuminoids form complexes with nontoxic metals likecalcium, zinc, magnesium, chromium, etc. Curcuminoids can be complexedwith metals using the following steps:

a) dissolving at least one curcuminoid in a mixture of methanol andacetone,

b) heating the resulting mixture to 40°-50° C.,

c) preparing a metal solution by dissolving a metal in a solvent,

d) adding the metal solution to the solution of curcuminoid to produce amixture,

e) adjusting the pH of the mixture to 7.5-9.5 to precipitate any metalcomplexes,

f) filtering any metal complexes which precipitate, and

g) drying the metal complexes.

An example of the above described process is given hereby for a calciumcomplex with curcuminoids. As discussed above, the present method issuitable to prepare other metal complexes with curcuminoids.

1. The 36.8 gm of curcuminoids is dissolved in a mixture of methanol andacetone.

2. The mixture is heated to 40°-50° C. for 1-2 hours.

3. Simultaneously, a solution of calcium chloride is prepared bydissolving 5.5 gm of calcium chloride in methanol. The solution isfiltered.

4. Calcium chloride solution is added to the solution of curcuminoidsand stirred for 2-3 hours.

5. The pH of the mixture is adjusted to 7.5-9.5 using ammonia toprecipitate the metal complexes.

6. The precipitate is filtered and washed with water and finally rinsedwith methanol.

7. The wet cake is dried at 70°-80° C.

8. The yield is approximately 25 gm of calcium complex withcurcuminoids.

The described method of complexation is novel for the following reasons:

the process complexes curcuminoids selectively;

the ratio of solvents used in the process is specific for completecomplexation;

the pH of the reaction is specific for quantitative isolation of thecomplex and stability of the isolated complex;

the purity of the complexed curcuminoids is higher than curcuminoidsalone, which may account for more potent biological activity.

The composition of the present invention can be administered alone, orit can be mixed with a pharmaceutically-acceptable carrier or diluentdepending on the mode of administration. Oral administration ispreferred, but parenteral and topical administration can be used. Fororal administration, the composition of this invention can be used inthe form of tablets, capsules, granules, powders, lozenges, syrups,elixirs, solutions, suspensions and the like, in accordance with thestandard pharmaceutical practice.

For parenteral administration, which includes intramuscular,intraperitoneal, subcutaneous and intravenous use, sterile solutions ofthe active ingredients are usually prepared, and the pH of the solutionsare suitably adjusted and buffered. For intravenous use, the totalconcentration of solutes should be controlled to render the preparationisotonic.

Carriers useful in formulating the preparations are commonly usedpharmaceutically acceptable non-toxic carriers such as gelatin, lactose,sodium citrate, salts of phosphoric acid, starch, magnesium stearate,sodium lauryl sulphate, talc, polyethylene glycol etc. The carrier maybe used with other additives such as diluents, binders, buffer agents,preservatives, sweetening agents, flavoring agents, glazes,disintegrators, coating agents, emulsifying agents, suspending agents,etc.

The daily dose of the preparation can be appropriately determined and isnot particularly limited. However, in most instances, an effectivedosage for an adult will be between 50-500 mg/three times per os.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of an assay to determine the effectiveness ofcurcuminoids in the prevention of free radical formation (preventionmode). Free radical formation is measured using the Rancimat method.

FIG. 2 shows the results of an assay to determine the effectiveness ofcurcuminoids in scavenging free radicals (intervention mode). Theability to scavenge free radicals is measured using the DPPH RadicalScavenging method.

FIG. 3 is an HPLC graph of the main components of the presentcomposition.

FIG. 4 shows the DPPH radical-scavenging ability of curcumin, tetrahydrocurcumin, and bis demethoxy curcumin.

FIG. 5 shows DPPH radical-scavenging ability of mixtures ofcurcuminoids.

FIG. 6 shows the effect of curcumin on NO₂ ⁻ production.

REFERENCES

1. Ho, C. T. , Chen, Q., Shi, H., Zhang, K. Q. and Rosen. R. T. (1992)"Antioxidative effect of polyphenol extract prepared from variousChinese tea", Preventive Med., 21:520-525 RANCIMAT METHOD!.

2. Yen, G. C., Duh, P. D. (1994) "Scavenging effect of methanolicextracts of peanut hulls on free-radical and active-oxygen species", J.Agric. Food Chem., 42:629-632 DPPH RADICAL SCAVENGING!.

We claim:
 1. A method for neutralizing free radicals and for preventingthe formation of free radicals in a patient, comprising administering toa patient in need of such treatment an effective amount of a compositioncomprising the following components: curcumin, demethoxy curcumin, andbis demethoxy curcumin, wherein said components are purifiedindividually or in combination and adjusted to the following ranges:75-81% curcumin, 15-19% demethoxy curcumin, and 2.2-6.5% bis demethoxycurcumin.
 2. The method according to claim 1, wherein said bis demethoxycurcumin is present in an amount less than 5%.
 3. A method for isolatingcurcuminoids, comprising the following steps:a) drying and powderingtumeric rhizomae to produce a resulting powder, b) extracting theresulting powder with a solvent at a temperature between 30°-60° C. toproduce an extract, c) concentrating said extract, d) lowering thetemperature of said concentrated extract to between 0°-15° C. tocrystallize any curcuminoids present, e) isolating any resultingcrystals, f) dissolving any isolated crystals of curcuminoids in asolvent at a temperature between 30°-50° C., g) lowering the temperatureof the dissolved curcuminoids to a temperature between 0°-15° C., and h)isolating any curcuminoids present.
 4. The method according to claim 3,wherein step b) is repeated at least once to produce additionalextracts.
 5. The method according to claim 3, wherein said extract isfiltered before concentration.
 6. The method according to claim 3,wherein said extract is concentrated by distillation under vacuum. 7.The method according to claim 3, wherein said crystals of curcuminoidsare dissolved in a C₁ -C₆ alkyl ketone solvent.
 8. The method accordingto claim 7, wherein said C₁ -C₆ alkyl ketone solvent is selected fromthe group consisting of acetone and methyl ketone.
 9. The methodaccording to claim 3, wherein said solvent is selected from the groupconsisting of ethylene dichloride, methylene dichloride and ethylacetate.
 10. A method for complexing curcuminoids with metals comprisingthe following steps:a) dissolving at least one curcuminoid in a mixtureof methanol and acetone, b) heating the resulting mixture to 40°-50° C.for 1-2 hours, c) preparing a metal solution by dissolving a metal in asolvent, d) adding the metal solution to the solution of curcuminoid toproduce a mixture, e) adjusting the pH of the mixture to 7.5-9.5 toprecipitate any metal complexes, f) filtering any metal complexes whichprecipitate, and g) drying the metal complexes.
 11. The method accordingto claim 10, wherein said solvent is methanol.
 12. The method accordingto claim 10, wherein said pH is adjusted using ammonia.
 13. The methodaccording to claim 10, wherein said metal complexes are washed withwater and rinsed with methanol after step f).
 14. The method accordingto claim 10, wherein said metal complexes are dried at a temperaturebetween 70°-80° C.
 15. The method according to claim 10, wherein saidmetal is calcium chloride.
 16. A method for protecting biologicaltissues from damage, comprising administering to a patient in need ofsuch treatment an effective amount of a composition comprising thefollowing components: curcumin, demethoxy curcumin, and bis demethoxycurcumin, wherein said components are purified individually or incombination and adjusted to the following ranges: 75-81% curcumin,15-19% demethoxy curcumin, and 2.2-6.5% bis demethoxy curcumin.
 17. Anbioprotectant composition, comprising the following components: 75-81%curcumin, 15-19% demethoxy curcumin, and 2.2-6.5% bis demethoxycurcumin, wherein said composition is produced by the following steps:a)drying and powdering tumeric rhizomae to produce a resulting powder, b)extracting the resulting powder with a solvent at a temperature between30°-60° C. to produce an extract, c) concentrating said extract, d)lowering the temperature of said concentrated extract to between 0°-15°C. to crystallize any curcuminoids present, e) isolating any resultingcrystals, f) dissolving any isolated crystals of curcuminoids in asolvent at a temperature between 30°-50° C., g) lowering the temperatureof the dissolved curcuminoids to a temperature between 0°-15° C., and h)isolating any curcuminoids present.